Immunoregulatory compounds and derivatives and methods of treating diseases therewith

ABSTRACT

Compounds are disclosed having the structure of Formula I:  
                 
 
     where R 1 , R 3 , and R 4  are independently hydrogen or C 1  to C 4  alkyl, and R 2  is:  
                 
 
     where R 5  is selected from the group consisting of hydrogen and C 1  to C 4  alkyl, or  
                 
 
     where R 6 , R 7  and R 8  are independently hydrogen or C 1  to C 4  alkyl; or the esters or pharmacologically acceptable salts thereof. Such compounds may be utilized for the prophylaxis or treatment of various diseases, particularly inflammatory conditions of the GI tract.  
     Methods of treating inflammatory conditions of the GI tract such as inflammatory bowel disease using compounds having the following formula are also disclosed:  
                 
 
     where R 9 , R 10  and R 11  are independently selected from the group consisting of hydrogen and C 1  to C 4  alkyl, and R 12  is selected from the group consisting of hydrogen and —C(O)R 13 , where R 13  is a C 1  to C 6  alkyl or an aryl group.

RELATED APPLICATIONS

[0001] This application is a divisional application of, and claimspriority to, U.S. patent application Ser. No. 09/942,464, filed Aug. 29,2001, allowed, which claims priority to U.S. Provisional Application No.60/228,683, filed Aug. 29, 2000, the disclosures of each of which areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

[0002] The present invention relates to immunoregulatory compounds andmethods of treating diseases therewith.

BACKGROUND OF THE INVENTION

[0003] Many people suffer from inflammatory bowel disease (IBD). IBD isa generic term used to refer to two inflammatory diseases, ulcerativecolitis and Crohn's disease. Ulcerative colitis is a chronicinflammatory disease of unknown etiology that affects various portionsof the gastrointestinal (GI) tract, particularly the lower GI tract, andmore particularly the colon and/or rectum. Crohn's disease is a seriousinflammatory disease of the GI tract. It predominates in the smallintestine (ileum) and the large intestine (colon). Various medicationsare being used to treat inflammatory bowel disease.

[0004] It is known to use mesalamine, 5-aminosalicylic acid (5-ASA) totreat ulcerative colitis. While mesalamine may be active in treatingulcerative colitis, it may be absorbed as it passes through the GItract. This absorption may adversely affect the amount of mesalaminethat reaches the lower GI tract, particularly the colon and rectum.

[0005] Various mesalamine formulations have been introduced in anattempt to protect mesalamine as it passes through the gut and the upperGI tract. One such formulation is a delayed-release formulation thatrelies on a pH-sensitive coating surrounding the mesalamine. The coatingallows the mesalamine to pass through the gut and upper GI tract withoutbeing absorbed so that the mesalamine reaches the target (i.e. the lowerGI tract, particularly the colon and/or rectum) intact. In anotherformulation, mesalamine microspheres surround a mesalamine core. Thisformulation releases mesalamine throughout the GI tract, rather thantargeting the colon specifically. It may be difficult to predict thebioavailability of the various mesalamine formulations when administeredto a wide variety of individuals. As a result, it may be difficult todetermine the proper dosage for a given individual.

[0006] It is also known to use sulfasalazine having the followingformula to treat ulcerative colitis.

[0007] However, sulfasalazine is metabolized in the body to formmesalamine (5-aminosalicylic acid (5-ASA)) and sulfapyridine. Severaladverse side affects have been noted from the use of sulfasalazineincluding nausea, vomiting, abdominal discomfort, and headache to namejust a few. These adverse side effects are usually attributed to theactivity of sulfapyridine in the GI tract, as well as that absorbed intothe system.

[0008] U.S. Pat. No. 4,412,992 to Chan proposes mesalamine derivatives.Unlike sulfalazine, the breakdown of these compounds in the intestinaltract may not give rise to undesirable metabolic products. In fact, thenon-mesalamine metabolic products may be innocuous.

[0009] Olsalazine having the following formula has been used to treatulcerative colitis.

[0010] In addition to being relatively expensive to make, olsalazine mayhave adverse side effects including diarrhea.

[0011] It is known to use azathioprine(6-(1-methyl-4-nitoimidazol-5-ylthio)purine) in the treatment ofinflammatory bowel disease. Azathioprine has the following chemicalstructure:

[0012] It is also known to use 6-mercaptopurine, a metabolite ofazathioprine, to treat inflammatory bowel disease. 6-mercaptopurine hasthe following chemical structure:

[0013] Methotrexate (L-4-amino-N¹⁰-methylpteroyl-glutamic acid) has alsobeen used to treat inflammatory bowel disease. Methotrexate has thefollowing chemical structure:

[0014] The polypeptide cyclosporine, which has traditionally been givento transplant patients to prevent organ rejection, has also been used totreat inflammatory bowel disease. The use of cyclosporine to treat IBDmay be limited, however, by the various side effects associated withthis medication. These side effects include high blood pressure, kidneydamage, tremors, headaches, seizures, excessive hair growth, excessivegum growth, confusion, coma, and gout.

SUMMARY OF THE INVENTION

[0015] According to embodiments of the present invention, compounds areprovided having the following formula:

[0016] where R¹, R³, and R⁴ are independently hydrogen or C₁ to C₄alkyl, and R² is:

[0017] where R⁵ is selected from the group consisting of hydrogen and C₁to C₄ alkyl, or

[0018] where R⁶, R⁷ and R⁸ are independently hydrogen or C₁ to C₄ alkyl,as well as the esters or pharmaceutically acceptable salts of suchcompounds. Pharmaceutical compositions including compounds according toembodiments of the present invention are also provided, as are methodsof treating inflammatory conditions with such compounds.

[0019] According to other embodiments of the present invention, methodsof treating an inflammatory condition of the GI tract in a subject inneed of such treatment include administering to the subject an effectiveamount of an active pharmaceutical ingredient that includes a compoundof Formula II:

[0020] where R⁹, R¹⁰ and R¹¹ are independently selected from the groupconsisting of hydrogen and C₁ to C₄ alkyl; and R¹² is selected from thegroup consisting of hydrogen and —C(O)R¹³, where R¹³ is a C₁ to C₆ alkylor an aryl group, or an ester or a pharmaceutically acceptable salt ofsuch compound, in admixture with a solid or liquid pharmaceuticaldiluent or carrier. The active pharmaceutical ingredient may furthercomprise a compound of Formula III:

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 illustrates embodiments of synthesis routes for compoundsof the present invention.

[0022]FIG. 2 illustrates embodiments of synthesis routes for compoundsof the present invention.

[0023]FIG. 3 illustrates the average reduction in colon:body weight [%BW] utilizing embodiments of the present invention (4-APAA/DNBS andMixture/DNBS) in comparison with results achieved by the prior art(5-ASA/DNBS) and control (Vehicle/DNBS).

[0024]FIG. 4 illustrates DNBS colitis adhesion scores achieved utilizingembodiments of the present invention (4-APAA/DNBS and Mixture/DNBS) incomparison with results achieved by the prior art (5-ASA/DNBS) andcontrol (Vehicle/DNBS and Vehicle/Sham).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The invention will now be described with respect to preferredembodiments described herein. It should be appreciated however thatthese embodiments are for the purpose of illustrating the invention, andare not to be construed as limiting the scope of the invention asdefined by the claims.

[0026] As used herein, the term “inflammatory bowel disease” includesulcerative colitis and Crohn's disease.

[0027] According to embodiments of the present invention, compounds areprovided having the following formula:

[0028] R¹, R³, and R⁴ are independently hydrogen or C₁ to C₄ alkyl.Preferably, R¹, R³, and R⁴ are independently selected from the groupconsisting of H, CH₃, CH₂CH₃, and CH(CH₃)₂. More preferably, R¹, R³, andR⁴ are H or CH₃.

[0029] R² is:

[0030] or

[0031] R⁵ is selected from the group consisting of hydrogen and C₁ to C₄alkyl. Preferably, R⁵ is selected from the group consisting of H, CH₃,CH₂CH₃, and CH(CH₃)₂. More preferably, R⁵ is H or CH₃ and, mostpreferably, R⁵ is H.

[0032] R⁶, R⁷ and R⁸ are independently hydrogen or C₁ to C₄ alkyl.Preferably, R⁶, R⁷ and R⁸ are independently selected from the groupconsisting of H, CH₃, CH₂CH₃, and CH(CH₃)₂. More preferably, R⁶, R⁷ andR⁸ are independently H or CH₃.

[0033] The compounds of the present invention may be made using knownstarting materials and reagents. For example, embodiments of synthesispaths may be illustrated as shown in FIGS. 1 and 2.

[0034] Compounds of the present invention may be utilized for theprophylaxis or treatment of various diseases, particularly inflammatoryconditions of the GI tract including, but not limited to, inflammatoryconditions of the mouth such as mucositis, infectious diseases (e.g.,viral, bacterial, and fungal diseases), and Crohn's disease;inflammatory conditions of the esophogas such as esophagitis, conditionsresulting from chemical injury (e.g., lye ingestion), gastroesophagealreflux disease, bile acid reflux, Barrett's esophogas, Crohn's disease,and esophageal stricture; inflammatory conditions of the stomach such asgastritis (e.g., Helicobacter pylori, acid-peptic disease and atrophicgastritis), peptic ulcer disease, pre-cancerous lesions of the stomach,non-ulcer dyspepsia, and Crohn's disease; inflammatory conditions of theintestine such as celiac disease, Crohn's disease, bacterial overgrowth,peptic ulcer disease, and fissures of the intestine; inflammatoryconditions of the colon such as Crohn's disease, ulcerative colitis,infectious colitis (e.g., pseudomembranous colitis such as clostridiumdifficile colitis, salmonella enteritis, shigella infections,yersiniosis, cryptosporidiosis, microsporidial infections, and viralinfections), radiation-induced colitis, colitis in the immunocompromisedhost (e.g., typhlitis), precancerous conditions of the colon (e.g.,dysplasia, inflammatory conditions of the bowel, and colonic polyps),proctitis, inflammation associated with hemorrhoids, proctalgia fugax,and rectal fissures; liver gallbladder and/or bilary tract conditionssuch as cholangitis, sclerosing cholangitis, primary bilary cirrhosis,and cholecystitis; and intestinal abscess. The compounds of the presentinvention may also be utilized in diagnosis of constituents, conditions,or disease states in biological systems or specimens, as well as fordiagnostic purposes in non-physiological systems. Furthermore, thecompounds of the present invention may have application in prophylaxisor treatment of condition(s) or disease state(s) in plant systems. Byway of example, the active component of the conjugate may haveinsecticidal, herbicidal, fungicidal, and/or pesticidal efficacyamenable to usage in various plant systems.

[0035] In some embodiments, compounds of the present invention maybreakdown in the intestinal tract to form the metabolic product ofFormula IV:

[0036] where R¹, R³ and R⁴ are as described above with reference toFormula I, and the metabolic product of Formula V:

[0037] The metabolic product of Formula IV may possess anti-inflammatoryactivity and/or immunoregulatory activity. The metabolic product ofFormula V may possess anti-inflammatory activity, and more particularlymay provide inhibition of prostaglandin synthetase I & II

[0038] In other embodiments, compounds of the present invention maybreakdown in the intestinal tract to form the metabolic product ofFormula IV and the metabolic product of Formula VI:

[0039] where R⁶, R⁷ and R⁸ are as described above with reference toFormula I. The metabolic product of Formula VI may possessanti-inflammatory activity and/or immunoregulatory activity.Accordingly, compounds of the present invention may provideimmunoregulatory activity. Compounds of the present invention may alsoprovide inhibition of prostaglandin synthetase I and II. Compounds ofthe present invention may be useful in treating various diseases,particularly ulcerative colitis, Crohn's disease and the like.

[0040] In therapeutic usage, the present invention contemplates a methodof treating an animal subject having or latently susceptible to anintestinal condition(s) or disease state(s) and in need of treatmenttherefor, comprising administering to such animal an effective amount ofa compound of the present invention that is therapeutically effectivefor said condition or disease state. Subjects to be treated by thecompounds of the present invention include both human and non-humananimal (e.g., bird, dog, cat, cow, horse) subjects, and are preferablymammalian subjects, and most preferably human subjects.

[0041] Depending on the specific condition or disease state to becombatted, animal subjects may be administered compounds of the presentinvention at any suitable therapeutically effective and safe dosage, asmay readily be determined within the skill of the art and without undueexperimentation. For example, compounds of the present invention may beadministered at a dosage between about 0.1 and 100 mg/kg, preferablybetween about 5 and 90 mg/kg, and more preferably between about 10 and80 mg/kg.

[0042] The compounds of the present invention may be administered per seas well as in the form of pharmaceutically acceptable esters, salts, andother physiologically functional derivatives thereof.

[0043] The present invention also contemplates pharmaceuticalformulations, both for veterinary and for human medical use, whichcomprise as the active pharmaceutical ingredient one or more compound(s)of the present invention. In such pharmaceutical and medicamentformulations, the active pharmaceutical ingredient preferably isutilized together with one or more pharmaceutically acceptablecarrier(s) therefor and optionally any other therapeutic ingredients.The carrier(s) must be pharmaceutically acceptable in the sense of beingcompatible with the other ingredients of the formulation and arepreferably not unduly deleterious to the recipient thereof. The activepharmaceutical ingredient is provided in an amount effective to achievethe desired pharmacological effect, as described above, and in aquantity appropriate to achieve the desired daily dose.

[0044] The formulations include those suitable for parenteral as well asnon-parenteral administration, and specific administration modalitiesinclude, but are not limited to, oral, rectal, buccal, topical, nasal,ophthalmic, subcutaneous, intramuscular, intravenous, transdermal,intrathecal, intra-articular, intra-arterial, sub-arachnoid, bronchial,lymphatic, vaginal, and intra-uterine administration. Formulationssuitable for oral and parenteral administration are preferred, withformulations suitable for oral administration most preferred.

[0045] When a compound of the present invention is utilized in aformulation comprising a liquid solution, the formulation advantageouslymay be administered orally or parenterally. When a compound of thepresent invention is employed in a liquid suspension formulation or as apowder in a biocompatible carrier formulation, the formulation may beadvantageously administered orally, rectally, or bronchially.

[0046] When a compound of the present invention is utilized directly inthe form of a powdered solid, the compound may advantageously beadministered orally. Alternatively, it may be administered bronchially,via nebulization of the powder in a carrier gas, to form a gaseousdispersion of the powder that is inspired by the patient from abreathing circuit comprising a suitable nebulizer device.

[0047] The formulations comprising a compound of the present inventionmay conveniently be presented in unit dosage forms and may be preparedby any of the methods well known in the art of pharmacy. Such methodsgenerally include the step of bringing a compound of the presentinvention into association with a carrier that constitutes one or moreaccessory ingredients. Typically, the formulations are prepared byuniformly and intimately bringing a compound of the present inventioninto association with a liquid carrier, a finely divided solid carrier,or both, and then, if necessary, shaping the product into dosage formsof the desired formulation.

[0048] Formulations of the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets, tablets, or lozenges, each containing a predetermined amount ofa compound of the present invention as a powder or granules; or asuspension in an aqueous liquor or a non-aqueous liquid, such as asyrup, an elixir, an emulsion, or a draught.

[0049] A tablet may be made by compression or molding, optionally withone or more accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine, with the active compound being in afree-flowing form such as a powder or granules which optionally is mixedwith a binder, disintegrant, lubricant, inert diluent, surface activeagent, or discharging agent. Molded tablets comprised of a mixture ofthe powdered active compound with a suitable carrier may be made bymolding in a suitable machine.

[0050] A syrup may be made by adding a compound of the present inventionto a concentrated aqueous solution of a sugar, for example sucrose, towhich may also be added any accessory ingredient(s). Such accessoryingredient(s) may include, for example, flavorings, suitablepreservatives, agents to retard crystallization of the sugar, and agentsto increase the solubility of any other ingredient, such as apolyhydroxy alcohol, for example glycerol or sorbitol.

[0051] Formulations suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of a compound of the presentinvention, which preferably is isotonic with the blood of the recipient(e.g., physiological saline solution). Such formulations may includesuspending agents and thickening agents or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. The formulations may be presented in unit-dose ormulti-dose form.

[0052] Nasal spray formulations comprise purified aqueous solutions of acompound of the present invention with preservative agents and isotonicagents. Such formulations are preferably adjusted to a pH and isotonicstate compatible with the nasal mucus membranes.

[0053] Formulations for rectal administration may be presented as asuppository with a suitable carrier such as cocoa butter, hydrogenatedfats, or hydrogenated fatty carboxylic acid.

[0054] Ophthalmic formulations are prepared by a similar method to thenasal spray, except that the pH and isotonic factors are preferablyadjusted to match that of the eye.

[0055] Topical formulations comprise a compound of the present inventiondissolved or suspended in one or more media, such as mineral oil,petroleum, polyhydroxy alcohols, or other bases used for topicalpharmaceutical formulations.

[0056] In addition to the aforementioned ingredients, the formulationsof this invention may further include one or more accessoryingredient(s) selected from diluents, buffers, flavoring agents,disintegrants, surface active agents, thickeners, lubricants,preservatives (including antioxidants), and the like.

[0057] Accordingly, compounds according to the present invention may beutilized for the prophylaxis or treatment of various diseases,particularly diseases of the GI tract including, but not limited to,inflammatory bowel disease.

[0058] In still other embodiments of the present invention, methods oftreating or preventing inflammatory bowel disease in a subject in needof such treatment or prevention include administering to the subject aneffective amount of an active pharmaceutical ingredient that includes acompound of Formula II:

[0059] where R⁹, R¹⁰ and R¹¹ are independently selected from the groupconsisting of hydrogen and C₁ to C₄ alkyl; and R¹² is selected from thegroup consisting of hydrogen and —C(O)R¹³, where R¹³ is a C₁ to C₆ alkylor an aryl group, or an ester or a pharmaceutically acceptable salt ofsuch compound, in admixture with a pharmaceutical diluent or carrier.

[0060] The active pharmaceutical ingredient may further comprise one ormore other medicaments including, but not limited to, anti-inflammatoryagents such as mesalamine, sulfasalazine, balsalazide, and olsalazine;immunomodulators such as azathioprine, 6-mercaptorpurine, cyclosporineand methotrexate; steroidal compounds such as corticosteroids; andantibiotics such as metronidazole and cirpofloxacin. The activepharmaceutical ingredient preferably further comprises mesalamine, thecompound of Formula III:

[0061] When the active pharmaceutical ingredient comprises compounds ofFormula II and Formula III, the compound of Formula II is preferablyfrom about 10 to 90 weight percent of the active pharmaceuticalingredient and is more preferably from about 40 to 60 weight percent ofthe active pharmaceutical ingredient. When the active pharmaceuticalingredient comprises compounds of Formula II and Formula III, the molarratio of the compound of Formula I to the compound of Formula II ispreferably between 1:10 and 10:1, and is more preferably between 1:2 and2:1.

[0062] Subjects to be treated by methods according to these embodimentsof the present invention include both human and non-human animal (e.g.,bird, dog, cat, cow, horse) subjects, and are preferably mammaliansubjects, and most preferably human subjects.

[0063] Depending on the specific condition or disease state to becombated, animal subjects may be administered the active pharmaceuticalingredient of the present invention at any suitable therapeuticallyeffective and safe dosage, as may readily be determined within the skillof the art and without undue experimentation. For example, the activepharmaceutical ingredient of the present invention may be administeredat a dosage between about 0.1 and 200 mg/kg, preferably between about 1and 90 mg/kg, and more preferably between about 10 and 80 mg/kg.

[0064] The present invention will now be described with reference to thefollowing examples. It should be appreciated that these examples are forthe purposes of illustrating aspects of the present invention, and donot limit the scope of the invention as defined by the claims.

EXAMPLES Examples 1 Through 4

[0065] Synthesis of Compounds of the Present Invention

[0066] Melting points were taken on a Laboratory Devices Mel-Temp IIcapillary melting point apparatus and are uncorrected. ¹HNMR spectrawere obtained on a Varian Unity 600 MHz spectrometer. Chemical shifts(δ) are reported as parts per million (ppm) relative to the internalstandard tetramethylsilane. Ultraviolet and visible spectra wereobtained with a Beckman DU 640i spectrophotometer. Infrared spectroscopywas obtained on a Nicolet Impact 410 and fast atom bombardment (FAB)mass spectroscopy data was obtained by M-Scan Inc. All reagents wereused as received from Aldrich Chemical Co.

Examples 1 and 2

[0067] Synthesis of 5-[4-(1-Carboxy-Ethyl)-Phenylazo]-2-Hydroxy-BenzoicAcid

Example 1

[0068] 2-(4-Amino-phenyl)-propionic acid p A 500-mL, oven dried,three-neck flask equipped with a stir bar, was charged with (R,S)2-(4-nitrophenyl)propionic acid (5.00 g, 25.6 mmol), absolute ethylalcohol (200 mL), and palladium (10 wt. % on activated carbon, 0.27 g,2.56 mmol). A hydrogen environment was introduced into the flask and themixture was then stirred at ambient temperature for 6 hours. The crudereaction mixture was filtered through Celite and the ethyl alcohol wasremoved under reduced pressure. The crude product was dried under vacuumovernight resulting in a light yellow solid (70% yield, 2.98 g): mp125-129° C., ¹H NMR (DMSO-d₆): δ1.24 (3H, s), 1.26 (3H, s), 3.41 (1H,s), 3.43 (2H, s), 6.46 (2H, d, J=7.6 Hz), 6.91 (2H, d, J=7.6 Hz); IR(KBr) 2596, 2189, 1630, 1581, 1441, 1375, 1277, 1192, 1052, 876 cm⁻¹;FAB-MS (NBA) m/z 165 (M+H)⁺.

Example 2

[0069] 5-[4-(1-Carboxy-Ethyl)-Phenylazo]-2-Hydroxy-Benzoic Acid

[0070] As prepared in the above procedure, 2-(4-amino-phenyl)-propionicacid (3.90 g. 23.6 mmol) dissolved in an aqueous HCl solution (75 mL,36.5-38.0% HCl in 8 mL H₂O) was placed in a 200-mL beaker and cooled to0° C. in an ice bath. When the solution was stabilized at 0° C., sodiumnitrite (1.79 g, 26.0 mmol) in water (2 mL) was added dropwise. Thetemperature was maintained at 0-5° C. and the resulting diazonium saltsolution stirred for 15 min.

[0071] While the diazonium salt solution stirred, an 800-mL beakerfitted with a stir bar, thermometer, and pH probe (Orion model 420A withOrion semimicro pH probe) was charged with salicylic acid, sodium salt(11.3 g, 20.8 mmol) dissolved in sodium hydroxide (4.25 g, 106 mmol) andH₂O (100 mL). Using an ice bath, the salicylic acid solution was cooledto 17° C. and the diazonium salt solution was slowly added in 10 mLportions. Throughout the addition, the pH was maintained at 13.2-13.3with the addition of aqueous sodium hydroxide, and the temperature waskept between 17-18° C. with the addition of ice. After the addition wascomplete, the resulting dark red solution was allowed to warm to ambienttemperature and stirring was continued for 90 min. Upon acidification topH 3.5 with concentrated HCl (˜20 mL, 36.5-38%), a dark red solidprecipitated and was collected by vacuum filtration.

[0072] The crude product (8.49 g, 27.0 mmol) was suspended in H₂O (300mL) and heated at 70° C. for 30 min. to remove excess salicylic acid.The suspension was cooled to 50° C. and a solid was collected by suctionfiltration. The collected solid was then purified by flashchromatography (SiO₂: ethyl acetate/hexanes, 1:1). The crude product(2.50 g. 7.95 mmol) in DMF (˜4.5 mL) was loaded and yellow coloredfractions were collected, combined, and concentrated under reducedpressure. After drying under vacuum, the purified product was obtainedas an orange solid in 55% yield (1.38 g): mp 147° C., ¹H NMR (DMSO-d₆):δ1.38 (3H, s), 1.39 (3H, s), 3.76 (1H, s), 3.78 (1H, s), 7.11 (1H,d,J=8.4 Hz), 7.46 (2H, d, J=7.8 Hz), 7.80 (2H, d, J=8.4 Hz), 8.03 (1H,d, J=9.0 Hz), 8.30 (1H, s); IR (KBr) 2973, 1921, 1708, 1652, 1577, 1477,1339, 1289, 1226, 1164, 1101, 1013, 857, 663 cm⁻¹; UV-Vis (MeOH)λ_(max)=355 nm, ε=23,700 mol⁻¹cm⁻¹L; FAB-MS (NBA) m/z 313 (M)⁻.

Example 3

[0073] Synthesis of 5-(4-Carboxymethyl-Phenylazo)-2-Hydroxy-Benzoic Acid[APAZA]

[0074] 4-Aminophenylacetic acid (10.0 g, 66.2 mmol) dissolved in anaqueous HCl solution (20 mL, 36.5-38.0% HCl in 200 mL H₂O) was placed ina 500-mL beaker and cooled to 0° C. in an ice bath. When the solutionwas stabilized at 0° C., sodium nitrite (5.02 g, 72.8 mmol) in water (50mL) was added slowly in 5 mL portions. The temperature was maintained at0-5° C. and the resulting diazonium salt solution stirred for 15 min.

[0075] While the diazonium salt solution stirred, a 2L beaker fittedwith a stir bar, thermometer, and pH probe (Orion model 420A with Orionsemimicro pH probe) was charged with salicylic acid, sodium salt (31.8g, 198 mmol) dissolved in sodium hydroxide (11.9 g, 230 mmol) and water(200 mL). Using an ice bath, the salicylic acid solution was cooled to17° C. and the diazonium salt solution was slowly added in 25 mLportions. Throughout the addition, the pH was maintained at 13.2-13.3with the addition of aqueous sodium hydroxide, and the temperature keptbetween 17-18° C. with the addition of ice. After the addition wascomplete, the resulting dark red solution was allowed to warm to ambienttemperature and stirring was continued for an additional 30 min. Uponacidification to pH 3 with concentrated HCl (˜50 mL, 36.5-38%), a brownsolid precipitated and was collected by suction filtration.

[0076] The crude product was purified by flash chromatography (SiO₂:ethyl acetate/hexanes, 1:1). On a column packed with 70-230 mesh, 60 Åsilica gel with BET surface area of ˜500 m²/g and pore volume of 0.75cm³/g, the crude product (11.5 g, 38.2 mmol) in DMF (˜12 mL) was loaded.Fractions were collected and combined based on color. The first band wasyellow in color and contained excess salicylic acid as well as traces ofthe desired product. The second band was orange and contained thedesired product, and the third band was red and contained unknownimpurities. All fractions were combined and concentrated under reducedpressure and dried under vacuum.

[0077] The purified product was obtained as an orange solid in 28% yield(2.75 g): mp 204° C.; ¹H NMR (DMSO-d₆) δ3.67 (2H, s), 7.11 (1H, d, J=9.0Hz), 7.44 (2H, d, J=8.4 Hz), 7.79 (2H, d, J=8.4 Hz), 8.02 (1H, d of d,J=2.4 Hz, 9.0 Hz), 8.29 (1H, s); IR (KBr) 3098, 1696, 1614, 1458, 1345,1195, 838 cm⁻¹; UV-Vis (MeOH) λ_(max)=350 nm, ε=25,700 mol⁻¹ cm⁻¹ L;positive FAB-MS (NBA) m/z 301 (M+H)⁺, negative FAB-MS(NBA) m/z 299 (M)⁻.

Example 4

[0078] Synthesis of 4-(4-Carboxymethyl-Phenylazo)-Phenylacetic Acid

[0079] 4-Aminophenylacetic acid (3.75 g, 24.8 mmol) was suspended inwater (75 mL) and concentrated hydrochloric acid (8 mL) was added. Thesolution was cooled to 0° C. in an ice bath with rapid stirring. Sodiumnitrite (1.80 g, 26.1 mmol) in water (20 mL) was added dropwise to the4-aminophenylacetic acid solution with rapid stirring. Care was taken tokeep the temperature between 0-5° C. at all times, especially during theNaNO₂ addition. The reaction was stirred for an additional 20 min. Inthe meantime, phenylacetic acid (10.1 g, 74.4 mmol) was dissolved in anaqueous NaOH solution (4.50 g, 113 mmol NaOH in 100 mL H₂O). Thesolution was vigorously stirred at 17° C. and at pH 13.3. The diazoniumsalt solution was added dropwise to the phenylacetic acid solution. Itis of utmost importance to keep the temperature of the phenylacetic acidsolution between 17-18° C. and the pH between 13.2-13.3 at all times,especially during the diazonium salt addition. The temperature wasregulated by the addition of ice and the pH regulated by the addition of8 M NaOH. After addition was complete, the solution was allowed to warmto room temperature and stirred for an additional 30 min. The reactionmixture was suction filtered to remove any undissolved particulates orunwanted side products. The filtrate was acidified with aqueous HCl (10mL conc. HCl in 20 mL H₂O) which produced a dark red precipitate. Theprecipitate was collected by suction filtration and washed several timeswith cold H₂O, until the filtrate was clear. The collected solid was airdried overnight to give the desired compound as a red solid in 37%yield: IR (KBr) 3030 (br), 1696, 1658, 1452, 1414, 1201, 850, 675 cm⁻¹FABMS m/z 299 (M+H)⁺, 320 (M+Na); ¹H NMR (DMSO-d₆) δ3.47 (s, 4H), 7.33(4H, d, J=8.1 Hz), 7.84 (4H, d, J=8.4 Hz).

Example 5

[0080] Metabolism of APAZA Following Oral Delivery

[0081] The degradation of Apaza(5-(4-carboxymethyl-phenylazo)-2-hydroxy-benzoic acid), a compound ofthe present invention, and sulfasalazine (used as a control; not part ofthe present invention) and the generation of their metabolites whenthese compounds were orally dosed to rats were measured to be able toconfirm that both Apaza and Sulfasalazine undergo bacterial azoreduction and yield their metabolites, 5-aminosalicylic acid (5-ASA) andsulfapyridine for sulfasalazine, 5-aminosalicylic acid (5-ASA) and4-aminophenyl acetic acid (4-APAA) for Apaza.

[0082] This experiment was performed to confirm that an azo compound,Apaza, undergoes bacterial reduction process and yields its metabolitesin in-vivo metabolism. The quantification of its metabolites was alsocarried out. Sulfasalazine, not part of the present invention, was usedas a control since similar azo bond cleavage by bacteria occurs with it,which results in 5-aminosalicylic acid and sulfapyridine as itsmetabolites. Both Apaza and sulfasalzine were degraded and theirmetabolites were produced as expected.

[0083] For urine, the parent compounds and their metabolites weredetected with day 1 collection only. The rest of the collections did notshow any compounds. For feces, compounds were detectable up to day 2collection.

[0084] Rats that were dosed with Apaza (rat 1, 2, and 3) showed Apaza,4-APAA, actarit, and acetylated 5-ASA in urine. Rats with sulfasalazinedosage (rat 4, 5, and 6) showed sulfasalazine, sulfapyridine, andacetylated 5-ASA in urine. Only acetylated 5-ASA was detected in fecesregardless of what compounds were given. 5-ASA was quickly converted toacetylated 5-ASA.

[0085] It is interesting to note that while sulfasalazine dosed ratsproduced their metabolites, 5-ASA (acetylated 5-ASA in this case) andsulfapyridine, in 1:1 ratio, rats with Apaza dosage produced 7 to 10times more of 4-APAA than acetylated 5-ASA.

[0086] It is believed that the majority of the ingested sulfasalazinetravels down the small intestine to the colon and undergoes bacterialazo reduction to liberate sulfapyridine and 5-ASA molecules. The resultsfrom this study confirm this belief and show that Apaza undergoes asimilar bacterial azo reduction.

[0087] A total of 8 rats were used for the experiment andmethylcellulose was used as a vehicle. The dosage amount was 100 mg/kgper rat. Three rats were dosed with Apaza and the other three rats weredosed with sulfasalazine. Two rats were used as a control and dosed withmethylcellulose. Both urine and feces were collected over 4 days andanalyzed by HPLC.

[0088] Urine was collected each day and 300 μL of aliquot from eachsample was centrifuged for 10 minutes at 5000 g. 80 μL of supernatantwas injected for analysis. Feces was also collected each day andhomogenized with 1:1 mixture of water and acetonitrile. This mixture wasthen centrifuged for 20 minutes at 5000 g. 80 μL of supernatant wasinjected for analysis.

[0089] A Waters 2690 HPLC was used for sample analysis as follows:Mobile phase programming: Gradient Mobile phase: A = Water + 0.1% TFA B= Acetonitrile + 0.1% TFA Flow rate: 1 mL/min. Column: Phenomenex MaxRP, 80 \, 4.6 mm × 250 mm PDA settings: Collected spectrum:210-400 nmExtracted chromatogram: 280 and/or other Run time/sample: Approximately50 mm. Flow % Mobile Phase % Mobile Phase Time (mL/minute) A B — 1 100 040 1 50 50 43 1 5 95 44 1 95 5 50 1 95 5

[0090] 5-ASA was quickly converted to acetylated 5-ASA. The same amountof acetylated 5-ASA was generated from both Apaza and sulfasalazine inurine. Acetylated 5-ASA and sulfapyridine were produced in 1:1 ratiofrom sulfasalazine dosed rat urine. Approximately 7 to 10 times more of4-APAA was produced than acetylated 5-ASA from Apaza dosed rat urine.Only acetylated 5-ASA was detected from feces regardless of dosedcompound. More acetylated 5-ASA was detected in feces than urine. Day 1Apaza Dosed Total Dosage Apaza 4APAA Actarit Acetylated 5ASA Urine (mg)(mg) (mg) (mg) (mg) Rat 1 22.0 0.48 3.456 0.0717 0.299 Rat 2 23.5 0.35463.177 0.422 Rat 3 22.5 0.4707 4.674 0.298 Sulfasalazine Dosed TotalDosage Sulfasalazine Sulfapyridine Acetylated 5ASA (mg) (mg) (mg) (mg)Rat 4 21 0.00882 0.337 0.288 Rat 5 22.5 0.01279 0.305 0.328 Rat 6 210.01092 0.41 0.39 Apaza Dosed Total Dosage Acetylated Stool (mg) 5ASA(mg) Rat 1 22 1.9254 Rat 2 23.5 1.9519 Rat 3 22.5 1.2437 SulfasalazineDosed Rat 4 21 1.2158 Rat 5 22.5 1.3708 Rat 6 21 0.9033 Day 2 ApazaDosed Total Dosage Acetylated Stool (mg) 5ASA (mg) Rat 1 22 0.2562 Rat 223.5 0.7755 Rat 3 22.5 0.1827 Sulfasalazine Dosed Rat 4 21 0.2 Rat 522.5 0.25 84 Rat 6 21 0.1458

Example 6

[0091] Biological Effects of Compounds of the Present Invention

[0092] The purpose of this study was to histologically evaluate andcompare the effects of three different active pharmaceutical ingredientsadministered intrarectally (twice daily for four days) to male Lewisrats following intrarectal administration of dinitrobenzene sulfonicacid (DNBS). DNBS induced colitis in rats according to an establishedexperimental model (Richard et al., 2000; Bertran et al., 1996; Blau etal., 2000; Kimura et al., 1998; Hogaboam et al., 1996). SHAM and DNBSgroups served as negative and positive controls, respectively. Thedistribution of animals to each group is presented in Table 1: TABLE 1GROUP NUMBER OF ANIMALS SHAM 6 DNBS 5 5-ASA 6 4-APAA 6 Mixture of 5-ASA4 and 4-APAA

[0093] Materials And Methods

[0094] Trimmed specimens of colon from 27 male rats were tested,including microtoming, and hematoxylin and eosin staining. The resulting27 slides (1 transverse section per slide) were examinedmicroscopically. Except for one rat from the SHAM group and one rat fromthe DNBS group, all slides had their labels taped over to facilitateblind reading. Lesions were graded on a scale of 1-5 (1=minimal; 2=mild;3=moderate; 4=moderately-severe; 5=severe).

[0095] Results

[0096] The principal histomorphologic change observed in the colonsections of all rats treated with DNBS (regardless of any additionaltreatment) was partial to full-thickness, full-length, coagulative-typenecrosis. Necrosis was not observed in the saline/methylcellulosetreated rats (SHAM group). In all cases, necrotic areas werecharacterized by a dramatic loss of cellular detail and stainingaffinity; in such areas only “ghost” outlines of the colonicarchitecture remained. Occasionally, segmental collapse or “dropout” ofan intestinal tissue layer was evident. Necrotic tissues were heavilyinvaded by mixed types of bacteria. In sections that were not completelynecrotic, the pattern of necrosis tended to be laminar, typicallyaffecting the mucosa and submucosa while sparing portions of themuscularis externa and/or aciventitia (serosa and adjacent mesentery).In these sections, a dense zone of karyorrhectic neutrophils divided thenecrotic inner layers from the less affected outer layers. Fibrinoidnecrotizing vasculitis of submucosal blood vessels was observed in allDNBS-treated rats. Vasculitis was observed in both necrotic andnon-necrotic regions, often accompanied by thrombosis (fibrinous,fibrinocellular, and/or bacterial thrombi), and minimal to moderatesubmucosal hemorrhage (with or without fibrin accumulation). Somehemorrhagic sites contained pigment-laden macrophages (siderophages—notseparately diagnosed). In all sections from DNBS-treated rats, theserosa and adjoining mesentery were expanded by mild to moderatelysevere fibrovascular proliferation (early granulation tissue). Sectionsfrom two rats (#4 and #11, Mixture of 5-ASA and 4-APAA group), eachcontained a single, short, sharply demarcated segment of non-necrotic,non-ulcerated mucosa. Changes within these comparatively unaffectedmucosal segments were limited to minimal to mild crypt epithelialhyperplasia, minimal crypt dilation, and minimal neutrophilicinfiltration.

[0097] Severity scoring of colonic necrosis was based upon the degree oftissue involvement; however, grade 5 (severe) was reserved for lesionsin which necrosis resulted in extensive tissue loss. Because the patternof necrosis often varied from section to section, the individualintestinal layers were scored separately. Generally, the averageseverity scores for necrosis were comparable among the four groups ofDNBS-treated rats (Table 2). The average score for mucosal necrosis inthe Mixture of 5-ASA and 4-APAA group was lower than scores in the othergroups of DNBS-treated rats due to the spared areas of mucosa in twoanimals from the Mixture of 5-ASA and 4-APAA group. TABLE 2 AverageTissue Necrosis Scores Mixture 5-ASA & Group SHAM DNBS 5-ASA 4-APAA4-APAA No. Animals (6) (5) (6) (6) (4) Mucosa 0.00 4.20 4.50 4.33 3.50Submucosa 0.00 4.20 4.17 4.00 4.25 Muscularis 0.00 3.60 3.5 3.17 3.00Adventitia 0.00 1.40 1.67 1.67 1.50

[0098] Summary

[0099] The principal histomorphologic change observed in the colonsections of all rats treated with DNBS (regardless of any additionaltreatment) was partial to full-thickness, full-length, coagulative-typenecrosis. Associated changes included massive bacterial invasion of thenecrotic tissue, fibrinoid necrotizing vasculitis with thrombosis andhemorrhage, and heavy neutrophilic infiltration. Necrosis was notobserved in the saline/methylcellulose-treated rats (SHAM group). Theseverity (extent) of necrosis was comparable among the four groups ofDNBS-treated rats (DNBS, 5-ASA, 4-APAA, and Mixture of 5-ASA and4-APAA), except that single segments of mucosa were comparatively sparedin 2/4 rats from the Mixture of 5-ASA and 4-APAA group.

Example 7

[0100] Anti-inflammatory Activity of Drug Mixture

[0101] Dinitrobenzene sulfonic acid (DNBS) colitis was induced (no etheranesthesia) in 4 groups of 6 Lewis rats each. One DNBS group was dosedwith vehicle (0.7% methyl cellulose) as well as an additional sham groupof 6 animals that received a saline enema instead of DNBS. Intrarectal(ir) dosing was performed in conscious animals b.i.d. for 4 days. Drugtreatments were as follows:

[0102] 5-aminosalicylic acid (5-ASA): 50 mg/kg

[0103] 4-aminophenylacetic acid (4-APAA): 49.5 mg/kg (equimolar to5-ASA)

[0104] Mixture: 5-ASA+4-APAA: 50 mg/kg+49.5 mg/kg

[0105] Drugs were suspended in the above mentioned vehicle and staffblinded to drug groups. Daily weights and diarrhea scores were recorded.On the 5th day post-irritant rats were sacrificed, laparotomiesperformed and scored for intestinal adhesions and strictures;colectomized and colon weights recorded, colons opened longitudinallyand inflammation/ulcerations scored.

[0106] Results illustrated in FIGS. 3 and 4 indicated that 5-ASA,4-APAA, and the mixture produce similar anti-inflammatory activity (˜31% reduction in colon:body weight [% BW]). The severity of inflammationapproached maximum. It is possible that the severity could be titratedby reduction of the DNBS dose and a small study was performed to testthis hypothesis. It is possible that with a milder insult there may beevidence of greater separation of treatment effects.

[0107] DNBS colitis was induced in 6 Lewis rats (3 at 30 and 3 at 15mg/rat DNBS) and allowed to develop for 5 days with no treatment inorder to citrate the severity of inflammation. Diarrhea was noted ondays 1-4 and the rats were sacrificed on day 5, scored, and colon:bodyweight determined. Results indicate that 15 mg/rat DNBS produces milderbut inconsistent inflammation compared to 30 mg. The 30 mg/kg DNBSresult was consistent with that seen previously.

[0108] The foregoing is illustrative of the present invention, and isnot to be construed as limiting thereof. The invention is defined by thefollowing claims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A method of treating an inflammatorycondition of the GI tract in a subject comprising administering to asubject in need of such treatment an effective amount of an activepharmaceutical ingredient comprising a compound of Formula II:

where R⁹, R¹⁰ and R¹¹ are independently selected from the groupconsisting of hydrogen and C₁ to C₄ alkyl; and R¹² is selected from thegroup consisting of hydrogen and —C(O)R¹³, where R¹³ is a C₁ to C₆ alkylor an aryl group; or an ester or a pharmaceutically acceptable salt ofsuch compound, in admixture with pharmaceutical diluent or carrier. 2.The method according to claim 1, wherein the active pharmaceuticalingredient further comprises a compound of Formula (III):

or an ester or a pharmaceutically acceptable salt thereof.
 3. The methodaccording to claim 1, wherein the compound of Formula II is from about10 to 90 weight percent of the active pharmaceutical ingredient.
 4. Themethod according to claim 1, wherein the molar ratio of the compound ofFormula II to the compound of Formula III is between about 1:10 and10:1.
 5. The method according to claim 1, wherein R⁹, R¹⁰, and R¹¹ areindependently selected from the group consisting of H, CH₃, CH₂CH₃, andCH(CH₃)₂.
 6. The method according to claim 1, wherein R⁹, R¹⁰, and R¹¹are independently selected from the group consisting of H and CH₃. 7.The method according to claim 1, wherein R¹² is hydrogen.
 8. The methodaccording to claim 1, wherein the compound of Formula II is4-aminophenylacetic acid.
 9. The method according to claim 1, whereinthe inflammatory condition of the GI tract is ulcerative colitis. 10.The method according to claim 1, wherein the inflammatory condition ofthe GI tract is Crohn's disease.
 11. The method according to claim 1,wherein the administering of an effective amount of the activepharmaceutical ingredient comprises orally administering to a subject inneed of such treatment an effective amount of the active pharmaceuticalingredient.
 12. The method according to claim 1, wherein theadministering of an effective amount of the active pharmaceuticalingredient comprises rectally administering to a subject in need of suchtreatment an effective amount of the active pharmaceutical ingredient.